This invention relates to a nucleic acid molecule encoding the catalytic subunit of a protein phosphatase (PP2AC-JD) that belongs to the PP2A family. The PP2AC-JD interacts with the phytochrome A, a primary photoreceptor in the light signal transduction in plants, in the photoperiodic control of flowering. The present invention also provides the methods and processes for generating transgenic higher plants transformed with said nucleic acid molecule to engineer flowering time of economically important crop plants.
The present invention is to provide a nucleic acid molecule encoding the catalytic subunit of a protein phosphatase 2A that dephosphorylates the phytochrome photoreceptors and has a role in the photoperiodic control of flowering time in plants, the methods and processes required for generating and analyzing biologically active polypeptides encoded by the nucleic acid molecule in enzymatic and biochemical assays, and the molecular ways for generating transgenic higher plants that exhibit delayed or accelerated flowering.
Plant flowering is regulated through complex interactions between intrinsic developmental factors and various environmental cues (Blxc3xa1zquez, 2000). The intrinsic factors include genes involved in the timing of flowering, flower meristem identity, and flower organ identity. Environmental cues that play critical roles in the flowering control are light, temperature, and water. Plant responses to these environmental cues appear in the forms of photoperiodism and vernalization in many cases (Izawa et al., 2000). Most of the signaling pathways that regulate flowering time and flower architecture have been elucidated by genetic analyses of mutant plants affected in a variety of aspects in flowering, and some of them, such as LEAFY and photoreceptors (Izawa et al., 2000; Reed et al., 2000; Weigel, et al., 1992), were molecular biologically and biochemically investigated in detail. A currently accepted model for the control of flowering in Arabidopsis depicts that there are four major signaling pathways, each mediated by light, gibberellic acid (GA), temperature, and circulating sucrose (Blxc3xa1zquez, 2000). Under the long day condition, light signals perceived by the photoreceptors trigger the activation of a facultative long day pathway in which the xe2x80x9cclock-relatedxe2x80x9d genes, such as ELF3, TOC1, LHY, CCA1, FKF1, and ZTL, are involved (Blxc3xa1zquez, 2000). However under the short day condition, flowering is exclusively regulated by light-independent signaling pathways that are mediated primarily by GA and temperature. The circulating sucrose, an indicator for the metabolic state of plants, also regulates flowering.
Light effects on the flowering are collectively called as photoperiodism, the lengths of alternating day and night. Based on the photoperiodic flowering responses, plants can be classified as two major categories; the short day plants that flower only in short days and the long day plants whose flowering occurs only in long days or activated by the long day condition, although some plants are day-neutral. At least two photoreceptors, the red and far-red light absorbing phytochromes and the blue light absorbing cryptochromes, are known to participate in the photoperiodic control of flowering in plants (Devlin and Kay, 2000; Mockler et al., 1999). The red light is perceived by the phytochrome B and eventually regulates the CONSTANS gene expression, whose gene product is a zinc finger transcription factor that regulates the transcription of the floral meristem identity genes (Blxc3xa1zquez, 2000). The far-red and blue lights are perceived by the phytochrome A and the cryptochromes, respectively, and regulate the expression of xe2x80x9cclockxe2x80x9d genes, such as ELF3, TOC1, LHY, CCA1, and ZTL, in the facultative pathway (Blxc3xa1zquez, 2000; Reed et al., 2000). The two light signaling pathways are eventually integrated by the CONSTANS transcription factor. Furthermore the light signals also interact with the GA-dependent pathway, especially under the short day condition (Blxc3xa1zquez, 2000).
Recent accumulating evidences suggest that the phytochromes and cryptochromes do not function independently but interact with each other in the flowering control (Guo et al., 2001; Mxc3xa1s et al., 2000; Mockler et al., 1999). The cryptochromes 1 and 2 (CRY1 and CRY2) are phosphorylated by the phytochrome kinases. The CRY1 and CRY2 interact with the phytochrome A (Ahmad et al., 1998), and the CRY2 with the phytochrome B in vitro. A Ca2+-binding protein, Arabidopsis SUB1, plays a role in the cryptochrome-phytochrome coaction (Guo et al., 2001). These interactions between the phytochromes and cryptochromes have also been functionally confirmed using mutant plants. These observations entail that a counter-acting partner to the phytochrome kinases, a protein phosphatase, would be involved in these interactions. In addition, based on the reversible phosphorylation of proteins in various signaling cascades in animals and plants (Palczewski et al., 1982; Stone et al., 1995), the presence of a protein phosphatase activity has been frequently implicated in the phytochrome-mediated light signal transduction during the photomorphogenesis, including the flowering control. However no such protein phosphatase gene has been identified so far.
In the present invention, we carried out a yeast two-hybrid screen using the C-terminal domain of the Arabidopsis phytochrome A as bait and a pea cDNA library to identify functional partners that specifically interact with the phytochrome A. One of the major positive clones isolated was a gene encoding the catalytic subunit of a protein phosphatase (designated PP2AC-JD in the invention) that belongs to the protein phosphatase 2A (PP2A) family (Virshup, 2000). The PP2AC-JD gene was exclusively expressed in the flower and stalk organs in a light-independent way. A recombinant PP2AC-JD protein expressed in E. coli expression system efficiently dephosphorylated the phosphorylated oat phytochrome A in the presence of Fe2+or Zn2+. The Pfr phytochrome was a better substrate for the PP2AC-JD than the Pr form. Transgenic Arabidopsis plants overexpressing the sense PP2A-JD gene flowered later than control plants, like a phytochrome A-deficient mutant plant. On the contrary, those with an anti-sense PP2AC-JD transgene flowered earlier with a time course similar to that observed in a phytochrome B-deficient mutant plant. These results indicate that the PP2AC-JD regulates the flowering time, possibly via the phytochrome-mediated light signal transduction pathway. This invention can be practically applied to control flowering time of higher plants economically important in agriculture and horticulture.
With rapidly accumulating technological information in recent years in the field of tissue culture and genetic transformation in plants, a gene of interest can now be routinely introduced into any desired plants with practical aims to enhance commercial value, yields, and environmental adaptability. For example, flowering plants can be engineered so that they flower earlier than control plants without any detrimental phenotypic effects. Furthermore the present invention can be applied to redistribute more metabolic nutrients into the vegetative organs than into the flowers by delaying flowering time, potentially resulting in improved productivity.
As used herein, the term xe2x80x9ceconomically important higher plantsxe2x80x9d refers to higher plants that are capable of photosynthesis and widely cultivated for commercial purpose. The term xe2x80x9cplant cellxe2x80x9d includes any cells derived from a higher plant, including differentiated as well as undifferentiated tissues, such as callus and plant seeds.
The present invention relates to nucleic acid molecules encoding the catalytic subunit of a protein phosphatase with structural and functional characteristics typical of the PP2A family. Such nucleic acid molecules preferentially encode a protein with the amino acid sequence as given in SEQ ID NO: 2 or fragments thereof that possess the enzymatic activity of the above-described protein phosphatase. The PP2AC-JD physically interacts with the C-terminal domain of the phytochrome A. It dephosphorylates the phytochrome A in vitro, preferentially the Pfr form, in the presence of Fe2+or Zn2+, a typical biochemical feature of the PP2A family (Virshup, 2000).
The present invention also relates to nucleic acid molecules that hybridize under high stringent conditions to a nucleic acid molecule as given in SEQ ID NO: 1. The term xe2x80x9chybridize under high stringent conditionsxe2x80x9d means that such nucleic acid molecules hybridize through complementary base pairing under conventional hybridization conditions.
Also, provided includes an uninterrupted gene sequence encoding the PP2AC-JD, a nucleic acid fragment that can be directly ligated into recombinant DNA constructs, and the PP2AC-JD expression vectors that can be readily used to transform cells of higher plants according to the present invention.
The present invention relates to a polypeptide or biologically active fragments of such a polypeptide encoded by said nucleic acid molecules to be used for enzymatic analysis. The protein phosphatase encoded by said nucleic acid molecules exhibits a dephosphorylating activity that is specific to the phytochrome dephosphorylation in a Fe2+or Zn2+-dependent way. Furthermore, the invention describes a polypeptide or biologically active fragments of such a polypeptide functionally expressed in bacterial cells. The polypeptide encoded by the above-described nucleic acid molecules shares common structural and functional properties with the catalytic subunit of the PP2A family.
Provided also are transgenic higher plants, especially flowering plants, that are readily accessible to the Agrobacterium-mediated transformation. Overexpression or anti-sense suppression of the PP2AC-JD gene results in delayed or early flowering, respectively. These phenotypic traits can be exploited in a way that higher plants of interest harboring the PP2AC-JD gene exhibit delayed or accelerated flowering, a very important commercial trait in horticulture and agriculture.
Therefore, the present invention provides: 1. Nucleic acid molecules encoding a polypeptide that dephosphorylates the phytochrome kinases in plants, comprising a nucleotide sequence as given in SEQ ID NO: 1. 2. Escherichia coli BL21 PP2AC-JD cells (KCTC 1003BP),=containing a nucleic acid molecule with the nucleotide sequence as given in SEQ ID NO:1, which is deposited at the Korean Collection for Type Cultures as International Depositary Authority on May 11, 2001 under Budapest Treaty.